1.1 Field of the Invention
The present invention relates generally to the field of virology, and in particular, to methods for preparing highly-purified, high-titer recombinant adeno-associated virus compositions. In certain embodiments, the invention concerns the use of equilibrium density centrifugation techniques, affinity chromatographic media, and in certain embodiments anion- and cation-exchange resins, to remove rAAV particles from solution and to prepare highly purified viral stocks for use in a variety of investigative, diagnostic and therapeutic regimens. Methods are also provided for purifying rAAVs from solution and for reducing the concentration of adenovirus in rAAV stocks.
1.2 Description of Related Art
1.2.1 Adeno-Associated Virus
Adeno-associated virus-2 (AAV) is a human parvovirus which can be propagated both as a lytic virus and as a provirus (Cukor et al., 1984; Hoggan et al., 1972). The viral genome consists of linear single-stranded DNA (Rose et al., 1969), 4679 bases long (Srivastava et al., 1983), flanked by inverted terminal repeats of 145 bases (Lusby et al., 1982). For lytic growth AAV requires co-infection with a helper virus. Either adenovirus (Ad; Atchinson et al., 1965; Hoggan, 1965; Parks et al., 1967) or herpes simplex virus (HSV; Buller et al., 1981) can supply helper function. Without helper, there is no evidence of AAV-specific replication or gene expression (Rose et al., 1972; Carter et al., 1983; Carter et al., 1983). When no helper is available, AAV can persist as an integrated provirus (Hoggan, 1965; Berns et al., 1975; Handa et al., 1977; Cheung et al., 1980; Berns et al., 1982).
Integration apparently involves recombination between AAV termini and host sequences and most of the AAV sequences remain intact in the provirus. The ability of AAV to integrate into host DNA is apparently an inherent strategy for insuring the survival of AAV sequences in the absence of the helper virus. When cells carrying an AAV provirus are subsequently superinfected with a helper, the integrated AAV genome is rescued and a productive lytic cycle occurs (Hoggan, 1965).
AAV sequences cloned into prokaryotic plasmids are infectious (Samulski et al., 1982). For example, when the wild type AAV/pBR322 plasmid, pSM620, is transfected into human cells in the presence of adenovirus, the AAV sequences are rescued from the plasmid and a normal AAV lytic cycle ensues (Samulski et al., 1982). This renders it possible to modify the AAV sequences in the recombinant plasmid and, then, to grow a viral stock of the mutant by transfecting the plasmid into human cells (Samulski et al., 1983; Hermonat et al., 1984). AAV contains at least three phenotypically distinct regions (Herrnonat et al., 1984). The rep region codes for one or more proteins that are required for DNA replication and for rescue from the recombinant plasmid, while the cap and lip regions appear to code for AAV capsid proteins and mutants within these regions are capable of DNA replication (Hermonat et al., 1984). It has been shown that the AAV termini are required for DNA replication (Samulski et al., 1983).
The construction of two E. coli hybrid plasmids, each of which contains the entire DNA genome of AAV, and the transfection of the recombinant DNAs into human cell lines in the presence of helper adenovirus to successfully rescue and replicate the AAV genome has been described (Laughlin et al., 1983; Tratschin et al., 1984a; 1984b).
1.2.2 Conventional Methods for Preparing Recombinant AAV
Recombinant adeno-associated virus (rAAV) has been demonstrated to be a useful vector for efficient and long-term gene transfer in a variety of tissues, including lung (Flotte, 1993), muscle (Kessler, 1996; Xiao and Samulski, 1996; Clark et al., 1997; Fisher et al., 1997), brain (Kaplitt, 1994; Klein, 1998) retina (Flannery, 1997; Lewin et al., 1998), and liver (Snyder, 1997). It has also been demonstrated to evade the immune response of the host by failing to transduce dendritic cells (Jooss et al., 1998). Phase I clinical trails are underway for cystic fibrosis rAAV-mediated gene therapy (Flotte et al., 1996; Wagner et al., 1998). Yet in spite of these promising developments one of the problems that remains to be solved is that vector production remains very laborious.
Currently rAAV is most often produced by co-transfection of rAAV vector plasmid and wt AAV helper plasmid into Ad-infected 293 cells (Hermonat and Muzyczka, 1984). Recent improvements in AAV helper design (Li et al., 1997) as well as construction of non-infectious mini-Ad plasmid helper (Grimm et al., 1998; Xiao et al., 1998; Salvetti, 1998) have eliminated the need for Ad infection, and made it possible to increase the yield of rAAV up to 105 particles per transfected cell in a crude lysate. Scalable methods of rAAV production that do not rely on DNA transfection have also been developed (Chiorini etal., 1995; Conway etal., 1997; Inoue and Russell, 1998; Clark etal., 1995). These methods, which generally involve the construction of producer cell lines and helper virus infection, are suitable for high-volume production.
However, little progress has been made on the downstream purification of rAAV. The conventional protocol involves the stepwise precipitation of rAAV using ammonium sulfate, followed by two or preferably, three rounds of CsCl density gradient centrifugation. Each round of CsCl centrifugation involves fractionation of the gradient and probing fractions for rAAV by dot-blot hybridization or by PCR(trademark) analysis. No only does it require two weeks to complete, but the current protocol often results in poor recovery of the vector and poor virus quality. The growing demand for different rAAV stocks often strains the limited capacities of vector production facilities. There is, therefore, a clear need for a protocol that will reduce the preparation time substantially without sacrificing the quality and/or purity of the final product.
In a first embodiment, the invention concerns a method of purifying a recombinant adeno-associated virus. In general, the method comprises centrifuging a sample containing or suspected of containing recombinant adeno-associated virus through at least a first iodixanol gradient, and collecting the purified virus or at least a first fraction comprising the recombinant adeno-associated virus, from the gradient. Preferably the gradient is a discontinuous gradient, although the inventors contemplate the formulation of continuous iodixanol gradients that also provide purification of rAAV compositions. In certain aspects of the invention, multiple iodixanol gradients, for example at least a second, at least a third and/or at least a fourth iodixanol gradient, are used to purify the recombinant adeno-associated virus.
In an exemplary discontinuous iodixanol gradient, the gradient comprises an about 15% iodixanol step, an about 25% iodixanol step, an about 40% iodixanol step, and an about 60% iodixanol step. Optionally, the gradient may contain steps having lower concentrations of iodixanol, and likewise, the gradient may contain steps that have higher concentrations of iodixanol. Naturally, the concentrations of each step do not need to be exact, but can vary slightly depending upon the particular formulation and preparation of each step. The inventors have shown that most rAAV particles will band in an iodixanol gradient at a level corresponding to a percentage of iodixanol approximately equal to 52%, although depending upon the number of viral particles loaded on the gradient and the volume and capacity of the gradient, the range of concentrations at which purified rAAV particles may be found may range on the order of from about 50% to about 53%, or from about 50% to about 54%, 55%, 56%, 57%, 58%, 59% and even up to and including about 60% iodixanol. Likewise, the range of concentrations at which the rAAV particles may be isolated following centrifugation may be on the order of from about 55% down to and including about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41% or about 40% or so iodixanol. Naturally, all concentrations in the range of from about 40% to about 60% are contemplated to be useful in recovering purified rAAV particles from the centrifuged gradient. As such, all intermediate concentrations including about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, and about 59% or so are contemplated to be useful in the practice of the present invention for recovering purified rAAV particles from the centrifuged gradient.
When step gradients are utilized, it is convenient to include in the gradient steps that encompass or xe2x80x9cbracketxe2x80x9d the range of optimal recovery of virus. For example, in a 25%/40%/60% step gradient, the 40% band comprises the virus, and this fraction is then removed for recovery of the virus composition. The design of both continuous and discontinuous gradients is well-known to those of skill in the art, and those having benefit of the present specification may readily prepare iodixanol gradients of sufficient capacity and range to isolate a band of purified rAAV particles from the gradient following centrifugation.
In certain embodiments, to improve the yield and/or recovery of virus particles from such a gradient, one may add to one or more steps of the gradient one or more salts to reduce or prevent aggregation of the virus and any cellular debris or proteins, polypeptides, etc. which may be present in the crude sample. In an exemplary embodiment, the inventors have shown that the addition of salt to the 15% iodixanol step in a discontinuous gradient improves the recovery of virus particles from an iodixanol gradient. As an example, the addition of NaCl to a final concentration of about 1 M in the 15% step was found by the inventors to be particularly advantageous in recovery of purified rAAV particles from the 40% step of such a gradient. While addition of one or more salts to one or more of the other steps in the gradient may be performed as required, in most instances, the inventors have shown that the presence of salt in other steps were either unnecessary or unwarranted. In situations where one or more salts are added to a layer which comprises the rAAV particles, following centrifugation it may be desirable to remove or reduce the concentration of salt in such a fraction prior to use of, or further purification of, the rAAV. Such removal may readily be achieved by dialysis, microconcentration, ultrafiltration, and the like.
In alternative embodiments, the inventors contemplate that the gradient may optionally comprise one or more additional compositions to permit farther, or enhanced purification of rAAV particles. Such compositions may include derivatives of iodixanol, iodixanol analogs, iodixanol-derived compounds, and/or compounds having centrifugation properties similar to, equal to, or superior to, iodixanol-alone compositions. Depending upon the particular composition added to the gradient, the relative position of the purified particles in the gradient may vary from that in which iodixanol alone is used (i.e. approximately 52% iodixanol), but such variance is readily overcome in the design of the gradient, and does not preclude the isolation of the rAAV from the particular density in the gradient where such virus particles are banded following centrifugation. Likewise, when one or more compositions are added to the iodixanol gradient, the centrifugation time, centrifugal force, and/or banding position within the gradient of the viral particles may be varied depending upon the particular application. Any such variations, improvements, or alterations in the composition of the iodixanol gradient are also contemplated to fall within the scope of this invention, and such modifications to the gradient will be apparent to those of skill in the art given the benefit of the teachings of the instant specification.
In a second embodiment, the invention relates to a method for purifying rAAV particles that comprises contacting a sample containing the virus with at least a first matrix that comprises heparin, under conditions effective and for a period of time sufficient to permit binding of the virus to the matrix, removing any unbound proteins or contaminants from the matrix, and then subsequently collecting or eluting the virus from the matrix. In exemplary embodiments, the matrix comprises heparin agarose type I or heparin agarose type II-S, although the inventors contemplate the use of any heparin composition or combinations thereof demonstrated to be effective in binding the rAAV, and thus removing it from a solution that is contacted with such a matrix. Preferably, the matrix is an affinity chromotographic medium, that may be comprised within a column, a syringe, a microfilter, or microaffinity column, or alternatively may be comprised within an HPLC affinity column. The matrix may be formed of any material suitable for the preparation of a heparin affinity matrix, and may, for example, be formulated as a resin, bead, agarose, acrylamide, glass, fiberglass, plastic, polyester, methacrylate, cellulose, sepharose, sephacryl, and/or the like. In fact, the inventors contemplate that the matrix may be fashioned out of any suitable material that forms a solid or semi solid support, and that permits the adsorption, ionic bonding, covalent linking, crosslinking, derivatization, or other attachment of a heparin moiety to the support matrix. Indeed, the art of affinity chromatographic medium preparation is sufficiently advanced so that a skilled artisan could readily prepare a suitable heparin affinity medium for use in purifying the rAAV particles using the methods disclosed herein. For example, the inventors have shown that an HPLC affinity column containing a crosslinked polyhydroxylated polymer derivatized with one or more heparin functional groups was useful in the purification of rAAV from a solution contacted with such a column.
Elution of the bound virus to the affinity column may be achieved in any manner convenient to the skilled practitioner, and may include, for example, the use of one or more elution buffers such as a salt buffer, to collect the virus from the column. In an exemplary embodiment, the inventors utilized a 1 M NaCl solution to elute the virus from the column. Prior to elution, the column comprising the bound virus may be washed with one or more washing or equilibrating buffers prior to elution of the virus from the column.
The use of an affinity column to purify rAAV particles may be used alone, or may be combined with the iodixanol gradient as described above to further increase the purification of the rAAV composition. One or more affinity columns may be utilized prior to the density gradient centrifugation purification method, and/or one or more affinity columns may be utilized after the purification through iodixanol gradients. In an exemplary embodiment, a cellular lysate containing rAAV particles is subjected to iodixanol centrifligation, and the fraction of the gradient containing the partially-purified rAAV is then contacted with at least one heparin affinity column to increase the total purity of the rAAV preparation.
Likewise, following either or both of the aforementioned purification methods, the rAAV composition obtained may be subjected to further purification, dialysis, concentration, and/or the like. In an exemplary embodiment, the partially-purified rAAV preparation may be further purified by contacting a fraction or sample containing or comprising recombinant adeno-associated virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species (proteins or other contaminants) with the hydrophobic matrix, and collecting the non-interacting virus from the hydrophobic matrix. Preferred are hydrophobic matrices that comprise phenyl groups, for example phenyl sepharose, phenyl sepharose 6 fast flow (low sub) or phenyl sepharose 6 (high sub). In certain embodiments, rAAV that has been partially purified by heparin affinity chromatography is further purified by hydrophobic interaction chromatography.
In other embodiments, the partially-purified rAAV preparation may be further purified by subjecting the viral sample to one or more cesium chloride equilibrium density gradients, and collecting from the gradient(s) the fraction(s) comprising the purified virus. The virus may then optionally be further purified by dialysis, microfiltration, microconcentration, and/or precipitation. Additionally, the virus may be further purified by contacting the virus with one or more ion exchange chromatography media, and eluting the virus from the media using one or more suitable elution buffers. Such an ion exchange chromatography medium may comprise a cation or an anion exchange medium. An exemplary cation exchange medium comprises at least one negatively-charged sulfonic group.
Contaminants that may be present in the sample containing the recombinant adeno-associated virus include, but are not limited to, viruses, such as adenovirus or herpes simplex virus, proteins, polypeptides, peptides, nucleic acids, cell extracts, growth medium, or combinations thereof. The methods of the present invention serve to reduce or eliminate one or more, or in certain embodiments all of the contaminants in a given recombinant adeno-associated virus sample. In preferred embodiments, the rAAV is about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, about 99.5% or more pure as judged by any of a variety of assays and analytical techniques that are known to those of skill in the art, including, but not limited to gel electrophoresis and staining and/or spectroscopy.
In certain embodiments, the invention provides methods for the preparation of highly-purified rAAV compositions comprising greater than about 1010 rAAV particles/ml. In exemplary embodiments, such methods have been demonstrated useful in the preparation of viral compositions comprising greater than about 1011, 1012, and even greater than about 1013 or 1014 particles/ml. In other embodiments, the invention provides methods for the preparation of rAAV compositions having a particle-to-infectivity ratio of less than about 100, and in certain aspects less than about 90, about 80, about 70, about 60, about 50 about 40, about 30, about 20 about 10, about 5, or in certain exemplary embodiments rAAV compositions having a particle-to-infectivity ratio of about 1.
The process for preparing highly-purified and/or highly-infectious viral preparations generally comprise the steps of centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient, collecting from the iodixanol gradient at least a first fraction comprising the recombinant adeno-associated virus, contacting the at least a first fraction comprising the recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of the virus to the matrix, removing non-bound species from the matrix, and eluting the virus from the matrix. Other methods for isolating rAAV provided by the present invention comprise the steps of centrifuging a sample containing or suspected of containing recombinant adeno-associated virus through an iodixanol gradient, collecting the purified virus from the gradient, contacting the virus collected from the gradient with a matrix comprising heparin, under conditions effective to permit binding of the virus to the matrix, collecting the virus from the matrix, subjecting the virus collected from the matrix to at least a first cesium chloride equilibrium density gradient, and collecting from the gradient a fraction comprising the highly-purified rAAV composition.
Additional methods of isolating a recombinant adeno-associated virus are also provided in the present invention. These methods generally comprises the steps of centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient, collecting from the iodixanol gradient at least a first fraction comprising the recombinant adeno-associated virus, contacting the at least a first fraction comprising the recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of the virus to the matrix, removing at least a first non-bound species from the matrix, eluting the virus from the matrix, contacting the eluted virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with the hydrophobic matrix, and collecting the non-interacting virus from the hydrophobic matrix.
Further methods generally comprise the steps of centrifuging a sample suspected of containing recombinant adeno-associated virus through an iodixanol gradient, collecting the purified virus from the gradient, contacting the virus collected from the gradient with a first matrix comprising heparin, under conditions effective to permit binding of the virus to the matrix, collecting the virus from the first matrix, contacting the virus collected from the first matrix with a second matrix comprising an anion exchange medium, and collecting from the second matrix a fraction comprising the purified virus.
In another embodiment, the invention provides a method of preparing recombinant adeno-associated virus. The method generally involves subjecting a sample suspected of containing recombinant adeno-associated virus to centrifugation through an iodixanol gradient, and collecting the virus from a fraction of the gradient corresponding to a concentration of iodixanol of about 40%. Such a gradient may be formed as described above, and may be prepared either as a continuous or a discontinuous gradient. In the case of discontinuous gradients, the gradient will preferably include at least an about 15% iodixanol step, an about 25% iodixanol step, an about 40% iodixanol step, and an about 60% iodixanol step, with the virus being isolatable from the 40% iodixanol step following centrifugation. Following recovery of the banded rAAV particles, the virus may be further purified using the heparin affinity chromatographic methods disclosed herein, and/or be optionally further purified via CsCl gradient centrifugation, anion exchange chromatography, cation exchange chromatography, affinity chromatography, or precipitation.
The invention also provides methods for reducing or eliminating adenovirus from a recombinant adeno-associated virus composition contaminated with adenovirus. The method generally comprises centrifuging a sample containing or suspected of containing both recombinant adeno-associated virus and adenovirus through one or more iodixanol gradients as described herein, and collecting the recombinant adeno-associated virus from the gradient. The concentration of adenovirus may be further reduced in such a sample by a number of methods, including, but not limited to, further purification on a heparin affinity column and/or a hydrophobic interaction column, by heating the sample, or alternatively, by anion exchange chromatography as described herein.
A method for reducing the concentration of adenovirus in a recombinant adeno-associated virus composition is also provided that generally involves centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient, collecting from the iodixanol gradient at least a first fraction comprising the recombinant adeno-associated virus, contacting the at least a first fraction comprising the recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of the virus to the matrix, removing any non-bound species from the matrix, and eluting the virus from the matrix.
A further aspect of the invention is the preparation of a high-titer rAAV composition. The method generally comprises the steps of: centrifuging a sample or rAAV through an iodixanol gradient, collecting the purified recombinant adeno-associated virus from the gradient; contacting the partially-purified recombinant adeno-associated virus collected from the gradient with a matrix comprising heparin, under conditions effective to permit binding of the recombinant adeno-associated virus to the matrix, and collecting the recombinant adeno-associated virus from the matrix. The purified rAAV composition eluted from the matrix may also be optionally further purified, such as in the case of the preparation of high-titer viral stocks, by contacting :the sample with a matrix comprising an anion exchange medium, under conditions effective to permit binding of the recombinant adeno-associated virus to the matrix, and collecting the purified recombinant adeno-associated virus from the matrix, preferably by elution.
The present invention thus also provides recombinant adeno-associated virus compositions, prepared by any one or more of the methods described herein. Generally, the invention provides at least a first recombinant adeno-associated virus composition, prepared by applying a sample containing recombinant adeno-associated virus to an iodixanol gradient, and collecting from the gradient at least a first fraction comprising the recombinant adeno-associated virus.
Also provided by the present invention are kits comprising combinations of the recombinant adeno-associated virus isolation media described herein. Generally, the kits comprise, in a suitable container, iodixanol and a matrix comprising heparin. In certain preferred aspects, the iodixanol is formulated as an iodixanol gradient. In other kits of the present invention, the matrix comprises heparin agarose type I or heparin agarose type II-S. Additional kits of the invention further comprise a hydrophobic matrix, such as a matrix comprising phenyl groups, exemplified by phenyl sepharose.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1. rAAV purification flow chart.
FIG. 2. Iodixanol step gradient for the purification of rAAV. Shown is a plot of the refractive index (vertical axis) of one ml-fractions (fraction number, horizontal axis) collected from the bottom of a tube after a 1 hour spin.
FIG. 3A and FIG. 3B. HPLC purification of the iodixanol fraction of rAAV-UF5, monitored at 231 nm. The absorbance at 231 nm (A231) is shown on the left vertical axis, time (min) is shown on the horizontal axis, and the ratio of diluent B (%B) is shown on the right vertical axis. FIG. 3A. POROS(copyright) HE/M chromatography. FIG. 3B. UNO(trademark) S1 cation exchange chromatography. The dotted line indicates the shape of the gradient. Elution time is shown in min above the respective peaks.